OA10338A - New nematophagous agent against nematodes of the meloidogyne genus - Google Patents

Abstract

Nematophage agent intended to combat nematodes of the Meloidogyne, Heterodera and Ditylenchus Myceliophagus genus, characterised in that it is selected within a group of six strains of Arthrobotrys conoides Drechsler (42A, 42A', 42B, 42Br, 42T and 42Vl, deposited at the national collection of cultures of micro-organism (Institut Pasteur) under the numbers I-1425 to I-1430, and application process consisting in incorporating the nematophage agent into the ground wherein the cultures are grown by means of grains which have been seeded previously with the inoculum of the nematophage agent.

Description

1 010338

TECHNICAL FIELD The present invention relates to a new agent for the control of nematodes of the genus Meloidogyne, and a method for controlling the proliferation of these nematodes by using new strains of the fungus Arthrohotrys. conoides Drechsler. The state of., The technique

It has long been known that nematodesphytophages cause considerable losses to 15 crops, estimated at 3 billion Swiss francs each year. The most prevalent and most damaging species in the world belong to the genus Meloidogyne. This genus, better known to farmers under the name of "gall-nematode", induces the formation of galls on the root system of attacked plants. These extremely polyphagous nematodes attack virtually all crops and are therefore responsible for yield reductions. catastrophic events of the order of 50 to 70%. In many areas, Heterodera carotae is a dangerous pest specific to carrot crops for which its bites trigger abnormal production of lateral rootlets and make the carrots non-commercializable. 50 Another nematode, Ditylenchus myceliophagus, can cause significant damage to agaric cultures in mushroom farms.

In order to combat these pests, farmers have several means which, in intensive cultivation, consist of calling for: - either preventive disinfection of the soil before cultivation using fumigants (Methyl Bromide, 2 010338

Chloropicrin, Dichloropropene. . ·) · These products, with a very wide spectrum of action, sterilize the soil and thus destroy the ecological balance of the environment. In addition, in light soil, their residues can pass into the water table, and in heavy soil their effectiveness is reduced by their bad diffusion either to curative treatments on certain cultures in place by means of systemic products conveyed by the sap (carbamates or organophosphates). This is for non-consumed productions such as flower crops or nurseries. These products are dangerous for both animals and humans because they pass into the plant leaving residual toxins. These two techniques act only on the first thirty centimeters of soil. Nematodes that can be found at a greater depth will invade the treated area again in the next culture. This involves a continual repetition of disinfections. In addition, because of their toxicity to humans, many chemical nematicides tend to be banned in many countries, as is the case in the Netherlands, Switzerland, Germany. SUMMARY OF THE INVENTION Therefore, the object of the invention is to use a natural agent for combating nematodes, which is not dangerous for humans and their environment. The main object of the invention is therefore a new nematophagous agent for combating the nematodes of the genus Meloidogyne, Heterodera and Ditylenchus myceliophagus, selected from six strains of the fungus Arthrobotrys conoiates Drechsler.

Another object of the invention is a method for controlling nematodes of the genus Meloidogyne, Heterodera and

Ditylenchus myceliophagus consisting in incorporating the fungus Arthrobotrys conoides Drechsler in the soil where the cultures were carried out by seed seeded in advance with the inoculum of the fungus.

BRIEF DESCRIPTION OF THE DRAWINGS The purposes, objects and features of the present invention are set forth in the following description for which the accompanying drawings illustrate certain aspects of the invention.

FIG. 1 schematically represents an uncidiophore and its conidia of the Arthrobotryscynosis strains according to the invention. FIG. 2 is a schematic representation of the traps formed by the Arthrobotrys strains according to the invention for capturing nematodes. FIG. 3 represents a table giving the result obtained with an electrophoresis gel used to differentiate the different strains of Arthrobotrysccr.aides.

Detailed description of the invention

Of the six strains to the Drosophler hemorrhoids that are subject to the invention come from different locations. The main criterion for verifying that the different strains belong to the same species is the leafure of conidia (reproductive organs of these fungi).

As illustrated in Figure 1, the conidia are grouped by 20 or 30 on the conidophore. The conidiophores can continue to develop beyond a first conidium and after a certain length of growth form a second group of conidia and thus destroy until the development of 5 to 6 additional groups.

Strain 42A is native of Fada N'Gourma in

In Burkina Faso, it consists of a mycelium composed of hyaline hyaline septa.

The conidiophores end with a head of about 20 conidia tight; on the aged cultures, they continue to develop and can produce successively 5 to 10 additional groups of conidia.

The conidia attenuate in a pronounced manner towards their base, they have an almost conical shape and are composed of two cells of different sizes, the total length of the conidium is on average 27 μm.

This strain has been deposited with the National Microorganism Culture Collection (CNCM) at the Institut Pasteur, where it was registered under number 1-1425.

The strain 42A 'is from Leguéma in Burkina

Faso.

The conidiophores terminate in a head formed from 3 to 20 conidia tight; on older cultures, they continue to grow and can produce 3 to 5 additional groups of conidia.

The conidia attenuate in a pronounced manner towards their base, they have an almost conical shape and are composed of two cells of different sizes, the total length of the conidium is on average 30 μm.

This strain was deposited with the CnCM (Institut Pasteur), where it was registered under number 1-1426.

Strain 42B is from the Baarn mycarium in the Netherlands.

On young cultures (less than one month old), cortiiophores are rare and end with a headform of 3 to 20 conidia; the formation of additional conidia is rare.

The conidia attenuate in a pronounced manner towards their base, they have an almost conical shape and are composed of two cells of different sizes, the total length of the conidium is on average 28 μm.

This strain was deposited with the CNCM (Institut Pasteur), where it was registered under the η * Σ-1427.

Strain 42Br is from Brazil. 5 01ü 33 8

The conidiophores are determined by a head formed of 10 to 20 conidia tight. They continue to develop and successively produce 5 to 10 additional groups of deconidies.

The conidia attenuate in a pronounced manner towards their base, they have an almost conical shape and are composed of two cells of different sizes, the total length of the conidium is on average 23 μm.

This strain has been deposited with. laCNCM \ Institut Pasteur), where it was registered under number 1-1428.

The strain 42T was harvested at Tours Indre-et-Loire (France).

The conidiophores end in a head formed of 10 to 20 conidia tight. On the aged cultures, they continue to develop and successively produce 3 to 5 additional groups of conidia.

The conidia attenuate in a pronounced manner towards their base, they have an almost conical shape and are composed of two cells of different sizes, the total length of the conidium is on average 28 pm.

This strain was the subject of a deposit with the CnCM (Institut Pasteur), where it was registered under number 1-1429.

The 42V1 strain was collected at Villeneuve LoubetAlpes-Maritimes (France).

The conidiophores end with a head formed of IC with 20 conidia tight. On the aged cultures, they continue to develop and successively produce 20 to 30 additional groups of conidia.

The conidia attenuate in a pronounced manner towards their base, they have an almost conical shape and are composed of two cells of different sizes, the total length of the conidium is on average 23 μm.

This strain was deposited with the CNCM (Institut Pasteur), where it was registered under the η * 1-1430. 6 010338

The mycelium of nematophagous fungi has the capacity to produce capture organs that trap nematodes. The traps of the mycelium of the six strains of Airthrobotrys conoides according to the invention are in the form of hyphalesans which become by anastomosis networks more or less complicated as illustrated in Figure 2.

Of the six strains studied, only strain 42A spontaneously forms traps. The other 5 form traps induced by the presence of nematodes in the culture medium. Whether spontaneous or induced, the traps consist of hyphal loops that become paranastomosis more or less complicated networks. The study of the genome of the six strains by the RAPD technique makes it possible to differentiate unambiguously the six layers by means of an oligonucleotide. The use of an electrophoresis gel has made it possible to identify the results of FIG. 3, in which the bands have been etched, and where the control serves to determine the size of the DNA fragments.

The different strains of Arthrobotrys conoides can be used industrially to control nematodes using the methods described hereinafter. The use of a culture medium based on seeds allows good conservation of the fungus. Cedernier takes refuge inside the seed and becomes less sensitive to attacks from the outside world. In addition, each seed spread on the ground is the starting point of a new mycelial colony.

The seeds are soaked for 24 hours in excess water before being sterilized. Then the seeds are seeded with the mushroom inoculum.

The best species to use as a support are those which allow to reach the maximum of growth in a minimum of time. A second criterion is the number of propagules per gram of support medium reached at the time of growth maximum, the propagation corresponding to a minimum fragment of mycelium or even a single secretion.

The species that can be used are among grasses, legumes, or oilseeds. Thus, it is possible to use hemp, sunflower and rice for which the time to reach the maximum of growth is 14 days. You can still use oats, oranges, barley, wheat, lentil, corn, canary grass, lesoja, beans, lupine, chickpeas, fava beans. Of the above species, those which make it possible to obtain a maximum number of propagules, ie approximately 3.times.10.sup.7 / g, are lupine, lentil, maize, bean, kelp, and chickpea. rice.

For a better distribution of the fungus, it is preferable to apply small weights to equal weight. For this reason and others, the seeds to be used preferably are those of canary grass, lentil and corn. In the case of maize, incorporation into the soil is satisfactory at the rate of 2 g per liter of soil. Because of the time required to obtain maximum growth, the fungus on its support is preferably incorporated into the soil about 15 days before planting.

The different strains of Arthrobotrys conoides can also be used as a lyophilized product. It is recalled here that the lyophilization is a sublimation desiccation technique of the ice resulting from solutions or suspensions of tissues, previously solidified by freezing. This technique commonly used for the conservation of microorganisms in the National Mycothec, involves a great respect of the operating conditions: low temperature to ensure the freezing of the product 35. reduced pressure to ensure the sublimation of the ice 8 01 0338 absence of interstitial liquid during the sublimation of the ice.

To fulfill these conditions, it suffices to induce an equilibrium between the pressure and the temperature at elevated temperature and to prevent the equilibrium from being restored by evacuation of the steam.

The freeze-drying of fungi can not be effected on the mycelium too sensitive to temperature and pressure variations, so the fungus must be made to yield conidia which can be assimilated to resins capable of being freeze-dried. This need to use conidia eliminating the use of any liquid medium, the fungus is grown on an agar medium based on soybean.

In order to eliminate the disadvantage presented by the presence of the agar-agar (agar) and by the weight of the culture medium (soy flour), a cellophane film-like sheet is interposed between the agar medium and the inoculum of the fungus. This paper, while separating the mushroom fungi, has the peculiarity of allowing the fungus to feed through the surface of the paper.

Once the mycelium has invaded the entire surface of the culture medium, the paper is removed and soaked in a liquid containing vitamins and microelements. In the liquid, the mycelium and conidia are detached from the cellophane paper. The set consisting of mycelium, conidia and vitamins and micro-elements is ensuitelyophiüsé. The presence of vitamins and micro-elements will allow a better recovery in the field.

The originality of this technique consists in the preliminary separation of the fungal fraction of the nutritive medium which has been used for its cultivation. Thus, the mushroom lyophilized is in the form of a powder that can be diluted in water, and therefore more easily used by the agrictalter. In this form, the fungus can be used in the same way as a chemical, mechanical spreading. In addition, this formulation has the advantage of considerably reducing the amounts of product to be spread on the ground and allows a better and longer preservation of the product. Because of the time required for maximum growth, the fungus Arthrobotrys conoides is preferably incorporated in the oleate to be treated about 15 days before planting. The efficacy of the different strains of Arthrobotrysconoides against Meloidogyne, Heterodera and Ditylenchus myceliophagus was first tested in in-vitro tests carried out in Petri dishes. To carry out these in vitro tests, the different strains of Arthrobotrys conoides were cultured on agar medium (corn meal agar: 1, agar-agar: 1) in petri dishes. When the fungus has invaded all the agar, 200 nematodes are deposited. aseptically in each box of mushrooms including control boxes containing only agar medium. After a lapse of time of 1h, 2h, 8h, and 24 hours, the agar contained in the boxes is reversed on a mesh of 100 μπι cells whose bottom is flush with the water of an underlying petri dish. This assembly allows the nematodes not trapped by the mushroom, to crossactively cross the screen to reach the water. The non-trapped nematodes are then counted under the magnifying glass.

In the case of Meloidogyne and Ditylenchusmyceliophagus, the six strains separate into two groups: strains A, A 'and B trap between 60% and 100% of nematodes in 24 hours, the other three strains being below this threshold.

In the case of Heterodera carotae, the six strains live between 50% and 75% of the nematodes in 24 hours.

It has also been observed that the development of the fungus was rapid (on average 1.5 cm per 24 hours).

It can therefore be used satisfactorily in practice as a nematophagous agent in garden and flower crops as well as in nursery haunches.

In the context of tests relating to the ecology of new predatory fungus strains of the same codes, it has been observed that they develop well in alkalis whose salinity does not exceed 3 g / l.

It has also been noted also that the predatory fungus invades the environment much more internally when it is introduced at a high dose, but this does not prevent it from colonizing the substrates in a satisfactory manner even at a dose of 10% of the volume of soil. The predictive activity of the various strains of Arthrobotrys conoides 42 has moreover been conformed by a certain number of practical application tests, some of which are given below as indicative. I. Test in tubes under greenhouse against Meloidoyoy The strains studied are divided into 2 categories, the trapping capacity of which is different: A, A 'and E mean average 80% of the nematodes in 24 hours; Br, T e ~. VI spends only 30% of Meloidogyne in 24 hours.

To carry out this test, we took the 2 25 most representative strains in each category, namely A and VI. This test was done in Ξ0 ml tubes containing 30 ml of soil. The field strains, grown on a crushed corn medium that had been sterilized, were incorporated into the soil at a dose of ICI g / m2. The tubes were treated as follows:

11 010338 Sol control not containing a predatory fungus A1 Soil containing the A strain, inoculation of the nematodes is carried out same day as the incorporation of the mushroom in the soil. VI 1 Soil containing the strain Vl, the inoculation of the nematodes is carried out same day as the incorporation of the mushroom in the ground. A15 Soil containing strain A, inoculation of nematodes takes place 15 days after incorporation of the fungus into the soil. VI Floc containing the strain Vl, inoculation of the nematodes takes place 15 days after incorporation of the fungus into the soil. Al ~ Vl The strain Vl is incorporated in the soil (in half dose: 50 g / m2) 15 days before the inoculation of the nematodes, and the strain A is added in half dose the same day as the nematodes.

On day 3, 30 Meloidogyne larvae are deposited in each tube. After 24 hours, the tubes of each series are taken out and their contents are analyzed in order to know the number of nematodes trapped. This manipulation is repeated daily until complete trapping of the nematodes.

The results obtained presented in the table below show that, regardless of the strain of Arthrobotrys 10 ccnoides, if the fungus is incorporated 15 days before being cultured (thus before the nematodes are activated by its host plant), the trapping varies from in the first days, but this difference diminishes rapidly. "15% average trapped nematode at D + 1% average trapped nematode at D + 2% average trapped nematode at D + 3 AI 46.14 66.67 90.28 VL 1 32.09 59.44 87.22 AT 5 63.12 80, 28 94.44 VL 15 61.8 86.11 93.06, A1 + V 15 44.92 88.89 95.06 12 II. Greenhouse trial in mini-pots with Meloidogyne 010338 hapla

The purpose of this test is to demonstrate the action of the fungus Arthrobotrys conoides on the nematode Meloidogyne hapla as well as to specify the quantity of product useful per m2. The 6 strains of Arthrobotrys conoides were tested with this nematode. The test was carried out in a mini-pot containing 300 g of soil, the plant used was a Tomato St Pierre, a variety very sensitive to nematodes.

Each pot was contaminated with 300 Meloidogyne haplasoit 100 nematodes per 100 g of soil, which corresponds to the upper threshold of an average infestation in the fields.

The results were read by root-to-eosin staining, a vital staining for counting the masses of first-generation eggs from the infective larvae that entered the root system.

Arrhroobotrys conoides is a predatory fungus20 which, in the absence of nematodes, can be maintained in the soil by digesting organic matter (nutritionsaprophage). Therefore, on the sterile soil of the plant, there is a sterile organic amendment (without micro-which allows a better development of these organisms) during the first 15 days of the test.

Calculation of the doses of treatment: one takes for base a nomo of propagules equivalent to, 108 propagules / m2 desol. However, for the 6 strains of Arthrobotrys conoides, this figure is reached on average with 15 g of crushed and autoclaved maize seed preparation. To allow some room for maneuver, the average dose common to the 5 strains was 40 g / m2, the second test dose was twice that, ie 80 g / m2. 35 replicates were made for each strain and for each dose without forgetting a control without a fungus. 13 * 1 3 / Results: for the average test dose common to the 6 strains and corresponding to 40 g / m2, the results are as follows: 010338 AA 'B Br T VI Control No. This mass of eggs / piant 65 150 145 150 125 123 163% mushroom efficacy 60% 8% 11% 8% 23% 24%

The best results were obtained with the A-A layer. The double test dose of 80 g / m 2 was as follows: 2A 2A 2B 2Br 2T 2V1 Control No. of egg mass / plant 32 116 133 146 94 77 163% efficacy of chamoicmon 80% 28% 18% 10% 42% 53%

As with the 40 g / m2 dose, the best results were obtained with strain A, but strain VI at this dose is also effective. For quick and reliable results, it is better to use strain A at the Sg / m2 rate when it is produced on a cooked crushed maize medium. 20 I1I_ Potted trial on carrot culture attacked by Heterodera carotae

This test was performed with strains A, B, T and VI. For this test, the different strains were cultured on a crushed cooked corn-based substrate. The quantities of mushroom brought were rigorously 010338 14

adjusted according to the strains so that there is always the same number of propagules per liter of soil (10a propagules / 100 1). For strains A and B, the inoculation dose was 60 g / 100 l of soil. For the T 5 strain the inoculation dose was 190 g / 100 1. Finally for the VI layer was used a dose of 8.0 g / 100 1 soil. A crop of unprocessed carrots with a control servings mushroom.

This test made it possible to count the cysts of Heterodera carotae from the generation of infective larvae. A B T Vl Control No. of cysts 39 .. 49 96 40 106% mushroom efficacy 63% 54% 9% 62%

Note that with Heterodera carotae as with Meloiddgyne hapla, the strains that give the best results are strains A and VI. IV. Greenhouse trial on tomato culture 20

This test was carried out in a greenhouse, in the agricultural sector with Arthrobotrys conoides 42A. It is intended to evaluate the dose of predatory fungus spread on the ground. For this test, the fungus is grown on a crushed corn medium. The content of this medium is 107propagules / ç.

The plot placed at our disposal is 120 m2.

It has been cut into three zones of 40 m2 each, which are separated from each other by planks in order to isolate them well, the first plot constitutes the control zone, it is followed by a zone treated with 50 g of WO preparation. 95 / 3-42 (19 PCT / FR95 / 00785 15 0 1 0338 42A per m2, then a parcel containing 100 g of preparation per m2.

Before setting up this test, a first analysis was made on soil taken between the culture in place (salad).

It was carried out by placing susceptible young seedlings (St Pierre variety) in this soil to reveal the presence of nematodes. One month after implantation, Lactophenol Blue-Cacton root staining (from GUIRAN, 1966) is performed. This coloration shows the juveniles of Meloidogyne having penetrated into the roots of the tomato.

We found 620 Meloidogyne larvae / 10 g urains, which corresponds to a medium infestation. This average infestation rate is ideal for testing the nematophagous fungus. Indeed, like any means of biological control, it acts slowly and can not regulate a very strong outbreak of nematodes on a single crop. Arthrobotrys conoides 42A was applied 15 days before planting tomato plants. This period of 15 days allows the predatory fungus to begin to colonize the soil and therefore be operational at the time of establishment of the crop. In fact, the tocnate plants emit radical exudates into the soil, which activates the hatching of Meloidogyne eggs and allows the young larvae to reach the roots.

The tomato plants are distributed in four rows, in the middle two rows (to avoid the effects of border, 10 feet of susceptible tomatoes (St Pierre variety) have been distributed, it is these plants which will be torn out for analysis.

The last samples were taken at the end of the cultivation. Susceptible plants were removed and the roots analyzed.

For this, the radical system once washed is milled in a 1% solution of chlorochemical calcium hypochlorite. This mash was passed on a series of detamis to remove plant tissue fragments. The eggs of Meloidogyne stemming from the root are collected on a sieve of 5 μιη and counted with the binocular loupe. They will constitute the infesting potential for the next crop.

Sample plot Plot at 50g / m2 Plot at 100g / m2 Number of eggs per 10g of root 44 581 25 838 17 725 10 It is good to specify certain points: - a female Heloidogyne can give, according to the climatic conditions, 300 to 3000 eggs, - an infestation is considered average when the number of larvae per 10 g of root is between 100 and 1000.

A significant decrease in the infesting potential (more than half) between the control plot and the plot treated with 100 m2 of Rrthrobotrys conoides42A is noted. 20

For plants available in clumps, buckets or containers, the mushroom-oatophage is directly incorporated into the root ball, the bucket or the container. Seeding by the predatory fungus 25 is carried out from the start in the pressed clumps which are used for the manufacture of market garden plants. In this way, it became apparent that within 20 to 30 days when the newly transplanted plants in the clods remain in a hot and humid greenhouse, the fungus 30 predates the time to grow and invade the entire root ball. which then behaves like an inoculum when it is set up in culture. This variant of the process according to the invention, applicable to the deliverable plants, in clumps, buckets, containers or the like, has proved to have the following decisive advantages: The soil usually used for making the 1 clods is a mixture rich in humus, at neutral pH, it is therefore particularly favorable to the development of Ariobrobotrys conoides 42. It has also been found that the root system of the plant is finds literally enveloped in mycelial felting which does not disturb its growth at all. The root system is therefore protected from attack by nematodes even if the root ball is planted in particularly contaminated soil. It thus appears that the young plant is well protected from the start of the vegetation, and it is generally when a plant is young that it fears the most attacks of nematodes.

The various experiments conducted have shown that the root ball constitutes an important inoculum, from which the mushroom very easily gains the solenvironment. The preparation of such lumps, buckets, containers or the like does not raise the slightest technical problem of use or manufacture of plants. It is sufficient to bring the granule into the mixture, for example using a hopper. Planting of the plants thus prepared is also quite usual. In summary, the process of the invention can be applied to all vegetable, flower and nursery crops, although some of these plants do not suffocate too much Meloidogyne nematodes. It can also be used on carrot crops that are susceptible to H. carotae, or against Ditylenchus myceliophagus in the case of the hgaricus bisporus mushroom. In the latter case, the nematophagous fungus can be added to the compost after pasteurization. κ

Claims (11)

18 010338 3 "CLAIMS $" 1. A nematophagous agent for combating "nematodes of the genus Meloidogyne, Heterodera and Ditylenchus, characterized in that it is chosen from the group of five layers of Arthrobotrys conoides Drechsler deposited in the National Collection of Cultures of Microorganisms (Pasteur Institute). ) respectively under the numbers: 1-1425, 1-1426, 1-1428, 1-1429 and 1-1430. 2. A method of controlling the nematodes of the genus Meloidogyne, Heterodera and Ditylenchus in cultures, incorporating in the soil where the cultures are grown, the nematophagous agent according to claim 1. 3. The method according to claim 2, wherein The nematophagous agent is incorporated into the soil by means of seed seeded in advance with the inoculum of said nematophagous agent. The method of claim 3, wherein said seeds are selected from the group consisting of aliphates, lentil and corn. 5. The method according to claim 4, wherein the nematophagic agent is pre-seeded on a milled corn-based medium containing at least 107 propagates of said agent per gram. 6. A method according to claim 4 or 5, wherein the nematophagous agent is seeded on a crushed, cooked and sterilized corn medium, said inoculated medium then being incorporated into the soil at a rate of 80 g / m 2. 7. The method according to claim 4 or 5, wherein the nematophagic agent is seeded on a crushed maize medium, cooked and sterilized, said inoculated medium then being incorporated in the soil at a rate of 2 g per liter of soil. The method of claim 2, wherein the nematophagic agent is previously lyophilized prior to being incorporated into the soil as a powder. 010338 19 9. The method of claim 3, wherein the nematophagus is pre-grown on a culture medium for the growth of conidia prior to freeze-drying. 10. A method according to claim 9, wherein a sheet of cellophane paper is interposed between the fruiting agent for growth and the culture medium so that the elements of said nematophagus containing the conidia are collected by soaking said cellophane sheet in liquid. 11. The method of claim 2, wherein the nematophagous agent is incorporated directly into the clod, scoop or container that is used to deliver nursery or market gardening plants. The method of any one of claims 2 to 11, wherein said nematophagous agent is incorporated in the soil approximately 15 days before planting.